Fuss, E. (Corresponding author)

1998
Forschungszentrum, Zentralbibliothek Jülich

Report No.: Juel-3500

Abstract: The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarum was fused to the mitochondrial signal sequence of Neurospora crassa 's ATPase subunit 9 and parts of bR were fused to the mitochondrial signal sequence of the subunit IV of the cytochrorne-c-oxidase of Saccharomyces cerevisiae. During expression of these geneconstructs in the cytoplasm of Schizosaccharomyces pombe only a small fraction of the fusion protein was imported into the mitochondria. The same constructs were tested for import into isolated mitochondria from Saccharomyces cerevisiae and Schizosaccharomyces pombe. This in vitro assay gave no evidence for an import of these proteins into the mitochondria. For the expression of bR in the mitochondrial matrix of Saccharomyces cerevisiae a gene for bR was adapted to the genetic code of these mitochondria. The gene was brought into the mitochondria via biolistic transformation and integrated into a complete mitochondrial DNA at the gene location for the subunit III of the cytochrome-c-oxidase, using homologous recombination. The transcription of the gene for bR in the mitochondrial matrix has been shown, but no translation product was detected. In summary, these experiments demonstrate that important problems about the import of very hydrophobic proteins into mitochondria are still unsolved. Transcription of bR in the matrix of yeast mitochondria is the first demonstrated transcription of a heterologous gene encoding a membrane protein in mitochondria.

Keyword(s): protein expression ; biological membrane ; bacteriorhodopsin ; gene expression ; ion pump ; gene transcription ; mitochondrion

1. Molekulare Biophysik (INB-2)